CN105758835B - A kind of homogeneous immunoassay POCT detection methods and the system using the detection method - Google Patents
A kind of homogeneous immunoassay POCT detection methods and the system using the detection method Download PDFInfo
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- CN105758835B CN105758835B CN201610288302.2A CN201610288302A CN105758835B CN 105758835 B CN105758835 B CN 105758835B CN 201610288302 A CN201610288302 A CN 201610288302A CN 105758835 B CN105758835 B CN 105758835B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
A kind of system the invention provides homogeneous immunoassay POCT detection methods and using the detection method.The homogeneous immunoassay POCT detection methods have the high sensitivity, high precision and wide scope of chemiluminescence immunoassay technology concurrently, while have the characteristics that POCT detection techniques are quick, portable；In addition, the homogeneous immunoassay POCT detection methods are due to being pure liquid phase detection, overcome the problem of next CV of NC film strips is higher, therefore detection precision is high, general CV is can be controlled within 5%, POCT detection techniques is met or exceeded the level of chemiluminescence immunoassay technology in terms of precision.The system using homogeneous immunoassay POCT detection methods, reagent card and POCT analyzers are separately designed, gather testing sample using reagent card, be easy to carry.
Description
Technical field
The invention belongs to immunoassay field, and in particular to a kind of homogeneous immunoassay POCT detection methods and use the inspection
The system of survey method.
Background technology
Existing chemiluminescence immunoassay technology is the mainstream technology of clinical examination analysis, with its high sensitivity, analysis
The features such as scope is wide receives an acclaim.It is primarily present following shortcoming：
A, instrument system is bulky, and floor space is big, simultaneously as test flux is big, reagent card is used with 100 tests
For whole unit, laboratory sample size is required；
B, instrument system and reagent price are expensive, and maintenance cost is high, is not suitable for basic medical unit；
C, equipment instrument is big, it is impossible to carries with into diagnosis and treatment scene；
D, chemiluminescence system can not typically use whole blood, limiting it makes mainly using serum and blood plasma as sample
Use scope.
Meanwhile a kind of clinical detection (bedside detection bedside carried out beside patient is newly risen in recent years
Testing real-time test (point-of-care testing) technology), abbreviation POCT detection techniques, POCT detection technique masters
Stream is fluorescent quantitation chromatography or collaurum, mainly wraps up the side that the fluorescent microsphere of fluorescent material or collaurum are analysed by film layer
Method progress immune detection examines technology soon.It is primarily present following shortcoming：
A, because both technologies mainly carry out release detection on NC films, because the CV of film in itself just has more than 5%,
Therefore the POCT detections CV of solid phase embrane method typically will be more than 10%, and CV% can even reach more than 20% during low concentration, inspection
Survey precision is very poor, and this brings huge challenge for quantitative analysis, quantitative for sensitivity requirement very high project such as cTnI
Just become extremely difficult.
B, the microballoon used in current POCT detection techniques is all the pure polystyrene surface of carboxyl modified, and the surface has very strong
Hydrophobicity, easily and nonspecific absorption occurs for albumen, therefore the poor anti jamming capability of detection, detection accuracy have dropped
It is low.
C, the POCT detection techniques of magnetic bead are currently based on due to being all heterogeneous system, it is necessary to the cleaning stream of complex designing
Journey.
D, latex determines the aggegation of a large amount of latex beadses by then passing through and light occurs and dissipates than the POCT detection techniques of turbid class
The change for penetrating signal detects antigen concentration, so the method for testing sensitivity is low, antibody consumption amount is big, and minute is long simultaneously
And much require that highly sensitive project can not be detected by latex agglutination.
E, latex lateral chromatography class POCT detection techniques, gold grain is replaced using colored latex, can visually carry out color
The depth is identified, miniature instrument can also be used to carry out signal acquisition, but due to being detected using CCD, color resolution is relatively low,
For the higher project of analyst coverage such as CRP, it may occur that situation about can not distinguish；
F, it is fast with reaction speed using new technique such as micro-fluidic chip type POCT detection techniques, sample requirement amount
The advantages that small, but also because reacting insufficient, the problem of causing detection sensitivity low.
The content of the invention
In order to solve above mentioned problem existing for prior art, the invention provides a kind of homogeneous immunoassay POCT detection sides
Method and the system using the detection method.The homogeneous immunoassay POCT detection methods have chemiluminescence immunoassay technology concurrently
High sensitivity, high precision and wide scope, while have the characteristics that POCT detection techniques are quick, portable；It is in addition, described homogeneous
Immunoassay POCT detection methods overcome the problem of next CV of NC film strips is higher, therefore detect essence due to being pure liquid phase detection
Density is high, and general CV is can be controlled within 5%, POCT detection techniques is met or exceeded chemical hair in terms of precision
The level of light immuno analytical method.
The system using homogeneous immunoassay POCT detection methods, reagent card and POCT analyzers are separately designed,
It is integrated to use, testing sample is gathered using reagent card, is easy to carry.
The technical solution adopted in the present invention is：A kind of homogeneous immunoassay POCT detection methods, comprise the following steps：
A, after testing sample is well mixed with oxygen supply microballoon reagent；It is well mixed again with by oxygen microballoon reagent；
B, laser irradiation is carried out to mixture in step A, while measures the luminous intensity that mixture is sent；
C, according to luminous intensity, the concentration of testing sample is conversed；
The oxygen supply microballoon reagent includes oxygen supply microballoon, it is described included by oxygen microballoon reagent it is micro- by oxygen microballoon, the oxygen supply
Ball and be polystyrene microsphere by oxygen microballoon, the oxygen supply microballoon is coated with hydrophilic aldehydedodextrans, described
Hydrophilic carboxyl glucan is coated with by oxygen microballoon；
The testing sample and the oxygen supply microballoon and it can occur specific reaction between by oxygen microballoon；
The wavelength of used laser is 680nm, and the wavelength of light is 520-620nm.
The principle of the homogeneous immunoassay POCT detection methods is：Biomolecule to be measured in testing sample is micro- with supplying oxygen
Ball and reacted to form immune complex by oxygen microballoon, this interaction oxygen supply microballoon and will can be furthered by oxygen microballoon, in laser
Under the irradiation of (wavelength 680nm), the oxygen in surrounding environment is converted into more active list by the sensitising agent supplied oxygen on microballoon
Body oxygen.Free oxygen is diffused to by oxygen microballoon, with being reacted by the chemiluminescence agent on oxygen microballoon, further have activated equally by oxygen
Luminophore on microballoon, it is allowed to send light, wavelength 520-620nm.The half-life period of free oxygen is 4 μ Sec, in the solution
Diffusion length is 200nm or so.If interaction is not present in biomolecule, free oxygen can not be diffused into by oxygen microballoon, then not
Have optical signal generation.Therefore the luminous intensity sent by measuring mixture, the biology point to be measured in testing sample can be calculated
The concentration of son.Also, because photooxidation microsphere composition surface covers one layer of hydrogel glucan, so as to greatly reduce system
In nonspecific absorption, improve detection stability and accuracy.
It should be noted that：Oxygen supply microballoon and be high polymer cladding sensitising agent (photooxidation dyestuff) by oxygen microballoon, and microballoon
There are aldehyde groups or carboxylic group in surface, for bioactive substances such as covalent bond antigen-antibodies.The sensitising agent is photooxidation
Dye substance methylene blue, rose-red, one kind or combination in phthalocyanine compound and chlorophyll A.
It is described oxygen supply microballoon and it is described by oxygen microballoon respectively coupling have biologic specificity material；
The biologic specificity material includes but is not limited to antigen, antibody, ProtinA, ProtinG and/or streptavidin；
Testing sample and be coupled at respectively the oxygen supply microballoon and by the biologic specificity material on oxygen microballoon can with occur it is special
Property reaction.
Native protein A (Protein A) is a kind of cell wall surface proteins for being found in staphylococcus aureus, natural egg
White G (Protein G) is a kind of cell surface protein for being isolated from G types or c-type streptococcus, and the two function is similar, main logical
Cross and interacted with the Fc areas of immunoglobulin (Ig), the IgG of most of mammals can be combined.
Certainly, biologic specificity material is not limited to antigen, antibody, ProtinA, ProtinG and/or streptavidin, only
, with reference to prior art, it can arbitrarily be designed to meet above-mentioned microballoon specificity under the technological thought of disclosure of the invention
With reference to specific biological molecules can as the present invention in biologic specificity material, just repeat no more herein.
The antibody includes first antibody and secondary antibody.
In the present invention, the testing sample includes but is not limited to body fluid sample, wherein body fluid sample include whole blood, serum,
Blood plasma, urine, saliva, amniotic fluid etc..
To improve the stability of the accuracy of final detection result and testing sample, preferable technical scheme is：Step A
In, first by the testing sample use diluted after, then with it is described oxygen supply microballoon reagent mix.
The dilution includes buffer solution, albumen, stabilizer, preservative etc..The dilution has what is diluted and buffer
Effect, improves the accuracy of final detection result and the stability of testing sample.
Under technological thought disclosed by the invention, the detection method of specific reaction includes but is not limited to：Double-antibody sandwich
Method, competition law, neutralize competition law, indirect method or prize law.
And when using some specific detection methods, except needing testing sample, oxygen supply microballoon reagent and by oxygen microballoon reagent
Outside, it is also necessary to extra reagent can just be smoothed out or can optimize progress, therefore preferable technical scheme is：In step A, first will
After the testing sample is well mixed with the first reagent, then with it is described oxygen supply microballoon reagent mix.It is it should be noted that of the invention
Described in the first reagent, be not specific to certain a kind of reagent, first reagent is some based on specific reaction in order to ensure
The reagent that the smooth or optimization of detection method is carried out and added, first reagent include but is not limited to：Biotinylated antigen
Or antibody；FITC mark antigen or antibody, resist biomolecule to be measured monoclonal antibody or polyclonal antibody, neutralize antigen,
Antigen.
Wherein, FITC is writing a Chinese character in simplified form for Fluorescein isothiocyanate isomer I, is biochemical reagents, mainly
For the fluorescent dye in fluorescent antibody technics, can be combined with various antibody proteins, the antibody with reference to after is not lost with necessarily resisting
The specificity that original combines, and there is strong green fluorescence in alkaline solution.
Discussed respectively with reference to specific detection method：
First, sandwich method
Sandwich method can continue to segment according to the difference of the immune complex pattern of formation：
1st, immune complex pattern is：It is micro- by oxygen to supply oxygen microballoon-streptavidin-Biotin-Antibody 1- Ag-Abs 2-
Ball, it is now, in the first reagent wells biotinylated antigen or antibody；Supply oxygen microballoon reagent wells in for coupling Streptavidin or
The oxygen supply microballoon of neutral Avidin；It is coupled antigen or antibody in by oxygen microballoon reagent wells by oxygen microballoon.
2nd, immune complex pattern is：Microballoon-anti-FITC-FITC- antibody 1- Ag-Abs 2- is supplied oxygen by oxygen microballoon, this
When, it is the antigen or antibody of FITC marks in the first reagent wells；Supply oxygen for coupling anti-FITC monoclonal antibody or more anti-in microballoon reagent wells
Oxygen supply microballoon；It is coupled antigen or antibody in by oxygen microballoon reagent wells by oxygen microballoon.
3rd, immune complex pattern is：Microballoon-secondary antibody-antibody 1- Ag-Abs 2- is supplied oxygen by oxygen microballoon, now,
It is the monoclonal antibody or polyclonal antibody for resisting biomolecule to be measured in first reagent wells；Supply oxygen in microballoon reagent wells as coupling the
The oxygen supply microballoon of two antibody；It is coupled antigen or antibody in by oxygen microballoon reagent wells by oxygen microballoon.
4th, certainly, sandwich method also has the situation without using the first reagent, as immune complex pattern is：Oxygen supply microballoon-anti-
Body 1- Ag-Abs 2- now, is supplied oxygen in microballoon reagent wells as the oxygen supply microballoon of coupled antibody by oxygen microballoon；Tried by oxygen microballoon
Agent Kong Zhongwei coupled antibodies by oxygen microballoon.
2nd, competition law, when biomolecule to be measured is antigen, immune complex pattern is：Oxygen supply microballoon-Ag-Ab-
By oxygen microballoon, now, supply oxygen in microballoon reagent wells as the oxygen supply microballoon of coupled antigen；It is coupled antibody in by oxygen microballoon reagent wells
By oxygen microballoon.
3rd, competition law is neutralized, immune complex pattern is：Microballoon-antibody 1- Ag-Abs 2- is supplied oxygen by oxygen microballoon, this
When, to neutralize antigen in the first reagent wells；Supply oxygen in microballoon reagent wells as the oxygen supply microballoon of coupled antibody；By oxygen microballoon reagent wells
In be coupled antibody by oxygen microballoon.
4th, indirect method, immune complex pattern are：Microballoon-Ag-Ab-secondary antibody-by oxygen microballoon is supplied oxygen, now,
Supply oxygen in microballoon reagent wells as the oxygen supply microballoon of coupled antigen；It is the micro- by oxygen of conjugated secondary antibody in by oxygen microballoon reagent wells
Ball.
5th, prize law, when biomolecule to be measured is IGM, immune complex pattern is：Oxygen supply microballoon-anti-μ antibody-anti-
Antigen-antibody-and by oxygen microballoon, it is now, in the first reagent wells antigen；Supply oxygen in microballoon reagent wells as the oxygen supply of the anti-μ antibody of coupling
Microballoon；It is coupled antibody in by oxygen microballoon reagent wells by oxygen microballoon.
IGM is immunoglobulin M (Immunoglobulin M) abbreviation.
Further to improve the accuracy of final detection result and the stability of testing sample, preferable technical scheme is：
In step A, the testing sample is first used into diluted, is well mixed after dilution with the first reagent, then with the oxygen supply
Microballoon reagent mixes.
Present invention also offers a kind of system using the homogeneous immunoassay POCT detection methods, including with the use of
Reagent card and POCT analyzers：
Testing sample hole, oxygen supply microballoon reagent wells are offered on the reagent card and by oxygen microballoon reagent wells；It is described to be measured
Sample well is used for containing testing sample, and the oxygen supply microballoon reagent wells are used for containing oxygen supply microballoon reagent, described to be tried by oxygen microballoon
Agent hole is used for containing by oxygen microballoon reagent；
Detection when, the reagent card is arranged in the POCT analyzers, the POCT analyzers include incubate module,
Reagent pipetting volume module, excitation module, optical signal detecting module and circuit control module；The incubation module, reagent pipetting volume mould
Block, excitation module, optical signal detecting module electrically connect with the circuit control module；
Under the control of circuit control module, the incubation module is used to adjust material in the reagent card and reagent card
Temperature, the reagent pipetting volume module are used to shift the material in the reagent card, and the excitation module is used to launch laser, institute
Optical signal detecting module is stated to be used to measure the luminous intensity that mixture is sent.
Under the control of circuit control module, the POCT analyzers can utilize prior art, support sandwich method, competition
Method, the reaction patterns such as competition law, indirect method or prize law are neutralized, while support Batch mode and random access patterns；
Batch mode can support multiple testing samples to carry out same analysis item detections, can also same testing sample carry out it is multiple
Project is analyzed simultaneously, can also support different testing samples, and disparity items is analyzed simultaneously, and so any one emergency treatment is to be measured
Sample can insert the analysis for carrying out any one project on menu at any time；Just repeat no more herein.
By the luminous intensity sent by measuring mixture, the dense of biomolecule to be measured in testing sample can be calculated
Degree.
Certainly, it is for the speed and accuracy that reduce the workload manually calculated and provide detection, preferable technical scheme：
The POCT analyzers also include software computing module, and the software computing module can carry out straight line, more according to being actually needed
Secondary (secondary, three times, four times, five times ...) equation, power function, index, 4 parameters or 5 parameter Log-Logistic fittings, and have
There are the functions such as batch calibration, and can be connected with hospital's LIS systems.
Flow using the system is：In the testing sample hole, oxygen supply microballoon reagent wells and by oxygen microballoon reagent wells
Testing sample, oxygen supply microballoon reagent are contained respectively and after by oxygen microballoon reagent, the reagent is placed in the POCT analyzers
In, the sample needle in the reagent pipetting volume module takes respective volume testing sample, adds oxygen supply microballoon reagent wells, by a timing
Between react after, continue to take certain volume mix after liquid add into by oxygen microballoon reagent wells, the excitation module is used to launch
Laser irradiates to by oxygen microballoon reagent wells, is reacted by certain time, what the optical signal detecting module measurement mixture was sent
Luminous intensity, by luminous intensity, calculate the concentration of the biomolecule to be measured in testing sample.
The accuracy of final detection result and the stability of testing sample are improved, preferable technical scheme is the reagent
It is further opened with diluting fluid apertures on card, the dilution fluid apertures is used for containing dilution；The testing sample hole, oxygen supply microballoon reagent
Hole, by oxygen microballoon reagent wells and dilution fluid apertures equal overlay film closed orifices, to ensure that wherein material is not contaminated；
For convenience of identifying and reading the information of testing sample, preferable technical scheme is to be additionally provided with bar on the reagent card
Shape code area, bar code is provided with the bar code area；The bar code is one-dimensional or Quick Response Code.
Corresponding, the POCT analyzers also include bar code scanning module, and the bar code scanning module, which is used to identify, to be read
Information in bar code.
The bar code scanning module supports IC-card scanning, printed bar code medium (paper or reagent card) scanning, and information is read
Enter using contact type scanning either non-contact scanning, its mode can be the modes such as infrared or radio frequency；Described information includes
But it is not limited to test and analyze project name, standard curve, reagent component, lot number, effect phase, manufacturer's information.
Further, the first reagent wells are further opened with the reagent card, first reagent wells are used for containing the first examination
Agent, the first reagent wells overlay film closed orifices.
It should be noted that when dilution and/or the first reagent in reaction system be present, the dilution and the first examination
The order of addition of agent is：First the testing sample is mixed with dilution and carries out dilution operation, after the completion of dilution, takes certain volume
Testing sample after dilution adds the first reagent wells, after mixing with the first reagent, continues to take the mixed liquid of certain volume
Into oxygen supply microballoon reagent wells, follow-up process is carried out.
It is described to be used as signal detection hole by oxygen microballoon reagent wells, it is described equal by the side wall of oxygen microballoon reagent wells and bottom
It is made of opaque material；The overlay film is covers high poly- plastic foil or pellosil, the poly- plastic foil of the height or pellosil and institute
There is biocompatibility with reagent.
Being shaped as the reagent card is circular, square or other geometries.The reagent card is single part reagent card.
The excitation module includes laser exciter；The optical signal detecting module includes photon detector (PMT).
In detection, the incubation module makes the reagent card and reagent card using modes such as metal bath, water-bath or oil baths
The temperature of interior material is 20-50 DEG C, and the material that the reagent pipetting volume module shifts every time is 1-500 μ L.
Preferably, the temperature of material is 35-45 DEG C in the reagent card and reagent card, and the reagent pipetting volume module turns every time
The material of shifting is 5-200 μ L.
Further, the temperature of material is 36-38 DEG C in the reagent card and reagent card, and the reagent pipetting volume module is each
The material of transfer is 20-100 μ L.
Beneficial effects of the present invention are：
1st, the homogeneous immunoassay POCT detection methods have the high sensitivity, high-precision of chemiluminescence immunoassay technology concurrently
Density and wide scope, while have the characteristics that POCT detection techniques are quick, portable；In addition, the homogeneous immunoassay POCT inspections
Survey method overcomes the problem of next CV of NC film strips is higher, therefore detection precision height, general CV can due to being pure liquid phase detection
Control makes POCT detection techniques to meet or exceed chemiluminescence immunoassay technology in terms of precision within 5%
Level.
2nd, the polystyrene microsphere used in the present invention, because one layer of hydrophilic aldehydedodextrans have been wrapped up on surface, because
This can greatly reduce non-specific adsorption, reduce the influence of system outer other environmental factors such as pH value and electrolyte etc., from
And the accuracy detected is greatly improved.
3rd, nanoscale microballoon is employed herein as immune solid phase, relative to traditional immunization solid phase, such as film, micropore
Plate, plastic bead etc., response area can greatly improve, and can effectively expand its detection range, reduce Hook effects.Relative to mesh
Preceding most widely used magnetic bead, is also more nearly homogeneous reaction, can make reaction speed faster, in similar heart function mark
Detection in, there is natural advantage；Simultaneously as need not wash, the time entirely analyzed can be greatly reduced, and favorably
In realize POCT automate.
4th, the homogeneous immunoassay POCT detection methods use oxygen supply microballoon and the system formed by oxygen microballoon, the confession
Oxygen microballoon and energy transmission can be carried out between by oxygen microballoon produce signal, and can be amplified step by step, and each supply oxygen micro-
Ball can release nearly 60,000 ion-oxygens, and release 10,000,000RLU per second is put by photomultiplier (PMT) to signal again
It is big to collect.Therefore the homogeneous immunoassay POCT detection methods have higher sensitivity for analysis, and required sample size is few,
So as to which the amount of antibody to be consumed is also less.From prepared by detection reagent cost it is cheaper, there is higher market competition
Power.
5th, present invention also offers a kind of system using the homogeneous immunoassay POCT detection methods, by reagent card and
POCT analyzers separately design, and gather testing sample using reagent card, are easy to carry；The POCT analyzers are simple to operate, real
Full-automation is showed, has avoided the interference of cleaning tape, there is the advantages of detection precision is high.
6th, the system is not because have wiper mechanism, and Instrument Design more minimizes and more stable, more portable, expansion
Instrument application field.
Brief description of the drawings
Fig. 1 is the immune complex schematic diagram in the present invention；
Fig. 2 is the top view of the reagent card in one embodiment in the present invention；
Fig. 3 is Fig. 2 front view.
In figure：1st, testing sample hole；2nd, fluid apertures is diluted；3rd, microballoon reagent wells are supplied oxygen；4th, by oxygen microballoon reagent wells；5th, bar shaped
Code area.
Embodiment
With reference to embodiment, present disclosure is further illustrated.It should be appreciated that the implementation of the present invention is not limited to
In the following examples, any formal accommodation and/or change made to the present invention fall within the scope of the present invention.
In the present invention, if not refering in particular to, all parts, percentage are unit of weight, and all equipment and raw material etc. are equal
It is commercially available or the industry is conventional.Method in following embodiments, it is the routine of this area unless otherwise instructed
Method.
Embodiment 1
As shown in figure 1, the invention provides a kind of homogeneous immunoassay POCT detection methods, comprise the following steps：
A, after testing sample is well mixed with oxygen supply microballoon reagent；It is well mixed again with by oxygen microballoon reagent；
B, laser irradiation is carried out to mixture in step A, while measures the luminous intensity that mixture is sent；
C, according to luminous intensity, the concentration of testing sample is conversed；
The oxygen supply microballoon reagent includes oxygen supply microballoon, it is described included by oxygen microballoon reagent it is micro- by oxygen microballoon, the oxygen supply
Ball and be polystyrene microsphere by oxygen microballoon, the oxygen supply microballoon is coated with hydrophilic aldehydedodextrans, described
Hydrophilic carboxyl glucan is coated with by oxygen microballoon；
Coupling has a biologic specificity material on the oxygen supply microballoon, it is described had by coupling on oxygen microballoon it is described by oxygen microballoon
Biologic specificity material；Testing sample with being coupled at the oxygen supply microballoon and by the biologic specificity material on oxygen microballoon respectively
Can be with generation specific reaction；
The wavelength of used laser is 680nm, and the wavelength of light is 520-620nm.
The principle of the homogeneous immunoassay POCT detection methods is：Biomolecule to be measured in testing sample is micro- with supplying oxygen
Ball and reacted to form immune complex by oxygen microballoon, this interaction oxygen supply microballoon and will can be furthered by oxygen microballoon, in laser
Under the irradiation of (wavelength 680nm), the oxygen in surrounding environment is converted into more active list by the sensitising agent supplied oxygen on microballoon
Body oxygen.Free oxygen is diffused to by oxygen microballoon, with being reacted by the chemiluminescence agent on oxygen microballoon, further have activated equally by oxygen
Luminophore on microballoon, it is allowed to send light, wavelength 520-620nm.The half-life period of free oxygen is 4 μ Sec, in the solution
Diffusion length is 200nm or so.If interaction is not present in biomolecule, free oxygen can not be diffused into by oxygen microballoon, then not
Have optical signal generation.Therefore the luminous intensity sent by measuring mixture, the biology point to be measured in testing sample can be calculated
The concentration of son.Also, because photooxidation microsphere composition surface covers one layer of hydrogel glucan, so as to greatly reduce system
In nonspecific absorption, improve detection stability and accuracy.
Embodiment 2
On the basis of embodiment 1, in step A, first by the testing sample use diluted after, then with the confession
Oxygen microballoon reagent mixes.
The dilution includes buffer solution, albumen, stabilizer, preservative etc..The dilution has what is diluted and buffer
Effect, improves the accuracy of final detection result and the stability of testing sample.
Embodiment 3
And when using some specific detection methods, except needing testing sample, oxygen supply microballoon reagent and by oxygen microballoon reagent
Outside, it is also necessary to extra reagent can just be smoothed out or can optimize progress, on the basis of embodiment 1 or embodiment 2, step
In A, after first the testing sample is well mixed with the first reagent, then with it is described oxygen supply microballoon reagent mix.
Embodiment 4
A kind of system using the homogeneous immunoassay POCT detection methods, including with the use of reagent card and POCT
Analyzer：
Testing sample hole, oxygen supply microballoon reagent wells are offered on the reagent card and by oxygen microballoon reagent wells；It is described to be measured
Sample well is used for containing testing sample, and the oxygen supply microballoon reagent wells are used for containing oxygen supply microballoon reagent, described to be tried by oxygen microballoon
Agent hole is used for containing by oxygen microballoon reagent；
Detection when, the reagent card is arranged in the POCT analyzers, the POCT analyzers include incubate module,
Reagent pipetting volume module, excitation module, optical signal detecting module and circuit control module；The incubation module, reagent pipetting volume mould
Block, excitation module, optical signal detecting module electrically connect with the circuit control module；
Under the control of circuit control module, the incubation module is used to adjust material in the reagent card and reagent card
Temperature, the reagent pipetting volume module are used to shift the material in the reagent card, and the excitation module is used to launch laser, institute
Optical signal detecting module is stated to be used to measure the luminous intensity that mixture is sent.Pass through the luminous intensity sent by measuring mixture, energy
Enough calculate the concentration of the biomolecule to be measured in testing sample.
In the present embodiment, the excitation module includes laser exciter；The optical signal detecting module includes photon
Detector (PMT).
Under the control of circuit control module, the POCT analyzers can utilize prior art, support sandwich method, competition
Method, the reaction patterns such as competition law, indirect method or prize law are neutralized, while support Batch mode and random access patterns；
Batch mode can support multiple testing samples to carry out same analysis item detections, can also same testing sample carry out it is multiple
Project is analyzed simultaneously, can also support different testing samples, and disparity items is analyzed simultaneously, and so any one emergency treatment is to be measured
Sample can insert the analysis for carrying out any one project on menu at any time；Just repeat no more herein.
Flow using the system is：In the testing sample hole, oxygen supply microballoon reagent wells and by oxygen microballoon reagent wells
Testing sample, oxygen supply microballoon reagent are contained respectively and after by oxygen microballoon reagent, the reagent is placed in the POCT analyzers
In, the sample needle in the reagent pipetting volume module takes respective volume testing sample, adds oxygen supply microballoon reagent wells, by a timing
Between react after, continue to take certain volume mix after liquid add into by oxygen microballoon reagent wells, the excitation module is used to launch
Laser irradiates to by oxygen microballoon reagent wells, is reacted by certain time, what the optical signal detecting module measurement mixture was sent
Luminous intensity, by luminous intensity, calculate the concentration of the biomolecule to be measured in testing sample.
The incubation module makes material in the reagent card and reagent card using modes such as metal bath, water-bath or oil baths
Temperature is 20-50 DEG C, and the material that the reagent pipetting volume module shifts every time is 1-500 μ L.
Embodiment 5
It is described for the speed and accuracy for reducing the workload manually calculated and providing detection on the basis of embodiment 4
POCT analyzers also include software computing module, and the software computing module can carry out straight line, repeatedly according to being actually needed
(secondary, three times, four times, five times ...) equation, power function, index, 4 parameters or 5 parameter Log-Logistic fittings, and have
The functions such as batch calibration, and can be connected with hospital's LIS systems.
As shown in Figures 2 and 3, the accuracy for raising final detection result and the stability of testing sample, the reagent
It is further opened with diluting fluid apertures on card, the dilution fluid apertures is used for containing dilution；The testing sample hole, oxygen supply microballoon reagent
Hole, by oxygen microballoon reagent wells and dilution fluid apertures equal overlay film closed orifices, to ensure that wherein material is not contaminated；The overlay film is
Pellosil is covered, the pellosil has biocompatibility with agents useful for same.
For convenience of identifying and reading the information of testing sample, bar code area, the bar code are additionally provided with the reagent card
Bar code is provided with area；The bar code is one-dimensional or Quick Response Code.
Corresponding, the POCT analyzers also include bar code scanning module, and the bar code scanning module, which is used to identify, to be read
Information in bar code.
Be shaped as circular or other geometries, the reagent card of the reagent card are single part reagent card.The examination
The temperature of material is 35-45 DEG C in agent card and reagent card, and the material that the reagent pipetting volume module shifts every time is 5-200 μ L.
Embodiment 6
On the basis of embodiment 5, it is described by oxygen microballoon reagent wells as signal detection hole, it is described by oxygen microballoon reagent wells
Side wall and bottom be made of opaque material；The overlay film to cover high poly- plastic foil, the poly- plastic foil of height with it is used
Reagent has biocompatibility.
The reagent card is shaped as square or other geometries, and the reagent card is single part reagent card.The examination
The temperature of material is 36-38 DEG C in agent card and reagent card, and the material that the reagent pipetting volume module shifts every time is 20-100 μ L.
It should be noted that when dilution and/or the first reagent in reaction system be present, the dilution and the first examination
The order of addition of agent is：First the testing sample is mixed with dilution and carries out dilution operation, after the completion of dilution, takes certain volume
Testing sample after dilution adds the first reagent wells, after mixing with the first reagent, continues to take the mixed liquid of certain volume
Into oxygen supply microballoon reagent wells, follow-up process is carried out.
Test example
With reference to concrete case, the homogeneous immunoassay POCT detection methods in the present invention are described further, by
The general technology scheme of immunoassay is can relate in method, following technology paths are only illustrated with sandwich method：
First, prepared by the covalent coupling of oxygen microballoon and antibody by oxygen microballoon reagent
1. by preparation amount measure 10mg be coated with carboxyl dextran hydrogel by oxygen microballoon in centrifuge tube,
10000rpm, centrifuge 60min.
2. abandoning supernatant, 2mg antibody, 50 μ l Tween-20 (50mg/ml) are added into precipitation, then add certain volume
0.05M MES pH=6.0, make by the final concentration of 10mg/ml of oxygen microballoon.
3. ultrasonic rapid mixing.
4. 50 μ l NaBH is added to centrifuge tube3CN (50mg/ml, 0.05M MES pH=6.0 preparations) is mixed, and 37 DEG C are put
36-48h is reacted in rotary mixer.
5. closing：1ml BSA (50mg/ml, 0.05M MES pH=6.0 preparations) is added, 37 DEG C are placed in rotary mixer
React 12-16h.
6. cleaning：With 0.05M MES buffer solution for cleaning three times.
7. be measured by sampling cleaning finish by the concentration of oxygen microballoon, particle diameter, signal value.
2nd, the covalent coupling for supplying oxygen microballoon and Avidin prepares oxygen supply microballoon reagent
1. supply oxygen microsphere suspension processing：Draw a certain amount of oxygen supply microballoon to centrifuge in high speed freezing centrifuge, discard
Supernatant, a certain amount of MES buffer solutions are added, ultrasound suspends again to particulate on ultrasonic cell disintegration instrument, adds the regulation of MES buffer solutions
Microballoon concentration is supplied oxygen to 100mg/ml.
2. avidin solution is prepared：A certain amount of Avidin (can also be Streptavidin or Neutravidin) is weighed,
Add MES buffer solutions to 8mg/ml.
3. mixing：Avidin the and MES buffer solutions for supplying oxygen microsphere suspension, 8mg/ml that will be handled well, with 2：5：1
Volume ratio is mixed, rapid to mix, and obtains reaction solution.
4. reaction：MES buffers 25mg/ml NaBH3CN solution, according to reaction solution 1：25 volume ratio adds
Enter, it is rapid to mix.37 DEG C of revolving reactions 48 hours.
5. closing：MES buffers 75mg/ml Gly solution and 25mg/ml NaBH3CN solution, according to it is anti-
Answer liquid 2：1：10 volume ratio is added in above-mentioned solution, is mixed, 37 DEG C of revolving reactions 2 hours.The BSA for adding 200mg/ml is molten
Liquid (MES buffer solutions), it is 5 with reaction solution volume ratio：8, rapid to mix, 37 DEG C of revolving reactions 16 hours.
6. cleaning：MES buffer solutions are added into completely reacted solution, high speed freezing centrifuge centrifugation, abandon supernatant, are added new
Fresh MES buffer solutions ultrasonic method suspends again, centrifuges again, so cleaning 3 times, is finally carried out with a small amount of by oxygen reagent buffer
Suspend, determine solid content, concentration will be adjusted by oxygen reagent buffer to 10mg/ml.
3rd, the preparation of biotin labelled antibodies
1. antibody is handled：Anti-HBe (can be antibody corresponding to any other analysis project) is dialysed in 0.1M
NaHCO3Solution, determine antibody concentration and adjust to 1mg/ml.
2. 16.17mg/ml Biotin solution is prepared with DMSO.
3. mark：Take the 1mg/mlAnti-HBe labelled antibodies handled well and the Biotin solution prepared, the two according to
10000：54 volume ratio is mixed, rapid to mix.2-8 DEG C stands reaction 12-16 hours.
4. dialysis：Completely reacted biotin labelled antibodies are dialysed in biotin labeling elution buffer (pH=8.00).
It is transferred to 5. the biotinylated antibody dialysed is suctioned out in clean centrifuge tube, antibody concentration is measured by sampling.By matter
Qualified biotin labelled antibodies concentration is examined to adjust to 0.5mg/ml.
4th, the preparation of quality-control product
Using NBCS as dilution, antigen sterling is diluted to the working solution of 2 various concentrations respectively, the working solution
As quality-control product Q1, Q2.Quality-control product Q1, Q2 to be detected is taken to carry out repeating to demarcate three times in our company's instrument system.Every time
10 holes are determined, and calculate population mean and SD, mean value ± 3SD is quality-control product concentration mensuration tolerance band.
5th, the preparation of calibration object
Antigen sterling is diluted to series concentration with calf serum (containing preservative), is calibrated with immunoassays with national standard
Afterwards, freezen protective is standby.During -20 DEG C of preservations, the term of validity 2 years.
Kit calibration object and the national standard of respective concentration carry out analysis measure simultaneously, with 4 parameters or other models
Fitting, it is desirable to which the absolute value of kit calibration object dose-response curve coefficient correlation (r) should be not less than 0.9900；Two simultaneously
Dose-response curve is not deviating significantly for parallel (t inspections)；Using national standard as standard, the actual measurement potency of kit calibration object
Ratio with demarcating potency should be between 0.90-1.10.
6th, upper machine testing
25 μ l serum samples are separately added into reacting hole, sequentially adding 25 μ l, (concentration is adjusted to 50 μ g/ by oxygen reagent
) and 25 μ l biotinylated antibodies reagents ml (concentration is adjusted to 4 μ g/ml).It is then placed in instrument and (likes that emerging biotechnology has by Chengdu
The liquid phase P OCT light-emitting appearances of limit company exploitation), operated according to the following steps automatically by instrument：Vibration, 37 DEG C incubate 5 minutes, then from
The dynamic coated microballoon reagent (concentration is adjusted to 60 μ g/ml) that supplies oxygen of 175 μ l Avidins that adds incubates 10 minutes for 37 DEG C afterwards.Instrument is certainly
Movable property life laser irradiation micropore calculates the photon amount that lighted per hole.
7th, examinations evaluation example
1st, withinrun precision is determined
Testing sample：The uneven quality controlled serum prepared according to the preparation of quality-control product " four, "
Process：Repeat detection 20 times
The criterion of outlier：≥3SD
Testing result such as following table：
Detect table 1
Note：
HBsAg：Hepatitis B virus surface antigen；
Anti-HBs：Hepatitis B surface antibody；
HBeAg：HBeAg；
Anti-HBs：Hepatitis B e antibody；
Anti-HBc：Hepatitis B virus core antibody；
L：Low-quality control；
H：High Quality Control.
As a result explain：This group experiment only one group of CV% more than 5%, is as a result equal to or exceeded chemiluminescence, say
Bright detection method has good precision.
2nd, sensitivity for analysis is determined
Sample：Value standard product
Process：Detection 20 times is repeated, obtains RLU (signal value)
Sensitivity：RLU substitutes into calibration curve
Testing result such as following table：
Detect table 2
| HBsAg | Anti-HBs | HBeAg | Anti-Hbe | Anti-HBc | |
| Mean | 610 | 901 | 162 | 66482 | 29511 |
| SD | 35 | 38 | 17 | 2870 | 2090 |
| CV% | 5.69 | 4.26 | 10.58 | 4.32 | 7.08 |
| Mean+2SD | 679 | 978 | 197 | 72223 | 33691 |
| Concentration unit | ng/mL | mIU/mL | PEI U/mL | PEI U/mL | PEI U/mL |
| Calculate sensitivity for analysis | 0.03 | 1.04 | 0.10 | 1.17 | 3.82 |
Note：
PEI U/mL, the non-WHO standard international unit of this unit, quantified entirely because five marks of HBV are not realized,
PEI U/mL formulate for German Paul-Ehrlich-Institute, are the most authoritative concentration quantitative unit in the current world；
As a result explain：The experiment of this group shows to obtain result, has uniformity with current international mainstream producer；
3rd, homogeneous immunoassay POCT detection methods of the present invention and the microballoon system of PerkinElmer companies detect
The application system of technology compares
By the microballoon system detection technique of PerkinElmer companies and described homogeneous immunoassay POCT detection methods according to
Upper example is parallel to carry out detection contrast
Testing result such as following table：
Detect table 3
As a result explain：The experiment of this group shows to obtain result, blank background, the present invention with PerkinElmer companies be it is suitable,
But it is demonstrated by more excellent precision and Geng Gao luminous efficiency in each concentration；
It should be noted last that the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although ginseng
The present invention is described in detail according to preferred embodiment, should be understood that the specific implementation that the foregoing is only the present invention
Mode, the protection domain being not intended to limit the present invention, within the spirit and principles of the invention, that is done any repaiies
Change, equivalent substitution, improvement etc., should be included in the scope of the protection.
Claims (10)
1. a kind of homogeneous immunoassay POCT detection methods, it is characterised in that comprise the following steps：
A, after testing sample is well mixed with oxygen supply microballoon reagent；It is well mixed again with by oxygen microballoon reagent；
B, laser irradiation is carried out to mixture in step A, while measures the luminous intensity that mixture is sent；
C, according to luminous intensity, the concentration of testing sample is conversed；
The oxygen supply microballoon reagent includes oxygen supply microballoon, described to be included by oxygen microballoon reagent by oxygen microballoon, the oxygen supply microballoon and
It is polystyrene microsphere by oxygen microballoon, the hydrophilic aldehydedodextrans that are coated with for supplying oxygen microballoon are described by oxygen
Microballoon is coated with hydrophilic carboxyl glucan；
The testing sample and the oxygen supply microballoon and it can occur specific reaction between by oxygen microballoon；
The wavelength of used laser is 680nm, and the wavelength of light is 520-620nm.
2. homogeneous immunoassay POCT detection methods according to claim 1, it is characterised in that the oxygen supply microballoon and institute
State by respectively coupling has biologic specificity material on oxygen microballoon；
The biologic specificity material includes but is not limited to antigen, antibody, ProtinA, ProtinG and/or streptavidin；It is to be measured
Sample and be coupled at respectively the oxygen supply microballoon and by the biologic specificity material on oxygen microballoon can with that specificity occurs is anti-
Should.
3. homogeneous immunoassay POCT detection methods according to claim 2, it is characterised in that the antibody includes first
Antibody and secondary antibody.
4. homogeneous immunoassay POCT detection methods according to claim 1, it is characterised in that in step A, first by described in
Testing sample use diluted after, then with it is described oxygen supply microballoon reagent mix.
5. homogeneous immunoassay POCT detection methods according to claim 1, it is characterised in that in step A, first by described in
After testing sample is well mixed with the first reagent, then with it is described oxygen supply microballoon reagent mix.
6. homogeneous immunoassay POCT detection methods according to claim 1, it is characterised in that in step A, first by described in
Testing sample uses diluted, is well mixed after dilution with the first reagent, then is mixed with the oxygen supply microballoon reagent.
A kind of 7. system that usage right requires any homogeneous immunoassay POCT detection methods of 1-6, it is characterised in that bag
Include the reagent card being used cooperatively and POCT analyzers：
Testing sample hole, oxygen supply microballoon reagent wells are offered on the reagent card and by oxygen microballoon reagent wells；The testing sample
Hole is used for containing testing sample, and the oxygen supply microballoon reagent wells are used for containing oxygen supply microballoon reagent, described by oxygen microballoon reagent wells
For containing by oxygen microballoon reagent；
In detection, the reagent card is arranged in the POCT analyzers, and the POCT analyzers include incubating module, reagent
It is loaded module, excitation module, optical signal detecting module and circuit control module；The incubation module, reagent pipetting volume module, swash
Light emitting module, optical signal detecting module electrically connect with the circuit control module；
Under the control of circuit control module, the temperature for incubating module and being used to adjust material in the reagent card and reagent card
Degree, the reagent pipetting volume module are used to shift the material in the reagent card, and the excitation module is used to launch laser, described
Optical signal detecting module is used to measure the luminous intensity that mixture is sent.
8. system according to claim 7, it is characterised in that it is further opened with diluting fluid apertures on the reagent card, it is described dilute
Fluid apertures is released to be used for containing dilution；The testing sample hole, supply oxygen microballoon reagent wells, be equal by oxygen microballoon reagent wells and dilution fluid apertures
Overlay film closed orifices；Bar code area is additionally provided with the reagent card, bar code is provided with the bar code area；
The POCT analyzers also include bar code scanning module, and the bar code scanning module is used to identify the letter read in bar code
Breath.
9. system according to claim 8, it is characterised in that the first reagent wells are further opened with the reagent card, it is described
First reagent wells are used for containing the first reagent, the first reagent wells overlay film closed orifices；It is described can by oxygen microballoon reagent wells
It is described to be made by the side wall of oxygen microballoon reagent wells and bottom of opaque material as signal detection hole；
In detection, the temperature of material is 20-50 DEG C in the reagent card and reagent card, and the reagent pipetting volume module shifts every time
Material be 1-500 μ L, the excitation module includes laser exciter；The optical signal detecting module includes photon detection
Device.
10. system according to claim 7, it is characterised in that in detection, material in the reagent card and reagent card
Temperature is 36-38 DEG C, and the material that the reagent pipetting volume module shifts every time is 20-100 μ L.
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